The present invention relates to a method of detecting neuromuscular disease in a patient, particularly Myoclonic Epilepsy and Ragged-Red Fiber disease (MERRF). More particularly, the invention relates to the detection of an adenine to guanidine transition mutation at nucleotide pair (np) 8344 in the human mitochondrial DNA (mtDNA), which alters the T.PSI.C loop of the tRNA.sup.Lys gene.
MERRF is a disease associated with uncontrolled myoclonic jerking and skeletal muscle deterioration involving accumulation of mitochondria in muscle cells, which stain red with Gomori modified trichrome and exhibit abnormal mitochondrial structures. The symptoms of the disease vary from mild central nervous system disturbances to deafness, status epilepticus, dementia, cardiomyopathy and respiratory failure.
MERRF has been shown to fulfill all of the criteria for a mitochondrial DNA (mtDNA) mutation. The disease is maternally inherited as is the mtDNA. MERRF is associated with defects in the mitochondrial oxidative phosphorylation (OXPHOS) Complexes I and IV whereas mtDNA encodes Complex I and IV subunits. The severity of the patient's skeletal muscle OXPHOS defect varies along the maternal lineage, consistent with the segregation of a heteroplasmic (mixed mutant and wild type) mtDNA mutation. Finally, as the patient's mitochondrial ATP generating capacity declines, tissues of the central nervous system, skeletal muscle and heart are progressively affected indicating that tissue-specific energetic thresholds are being traversed. However, detailed restriction analysis of MERRF mtDNA has failed to reveal any evidence of insertion-deletion or rearrangement mutations (Wallace et al., Cell, 55:601-610 (1988)). Hence, MERRF must be the product of a heteroplasmic mtDNa point mutation.
The mtDNA codes for 7 subunits (NDI, 2, 3, 4L, 4, 5, 6) of respiratory Complex I (NAHD:ubiquinone oxidoreductase), 3 subunits (COI, II and III) of Complex IV (cytochrome c oxidase), 2 subunits (ATPase 6 and 8) of Complex V (ATP synthase) and one subunit (cyt b) of Complex III (ubiquinol:cytochrome c oxidoreductase). In addition, the mtDNA encodes a large and small rRNA gene and a set of 22 tRNAs, including a single tRNA.sup.Lys, which recognizes the codons AAA and AAG (Anderson et al., Nature, 290:457-465 (1981); Shoffner and Wallace, Advances in Human Genetics, 19:267-330 (1990)). The 13 mtDNA polypeptides are translated on chloramphenicol-sensitive mitochondrial ribosomes and can be differentially labeled by incubating cells in medium containing .sup.35 S-methionine plus the cytosolic ribosome inhibitor emetine. All the mitochondrial translation products have been assigned to mtDNA genes, and the largest and most numerous of these are subunits of Complexes I and IV (Wallace et al., Am. J. Hum. Genet., 38:461-481 (1986); Chomyn et al., Science, 234:614-618 (1986)). In cultured cells, mutations in the mitochondrial large rRNA gene have been isolated which impart chloramphenicol resistance (Wallace, "Cytoplasmic Inheritance of Chloramphenicol Resistance in Mammalian Cells", Techniques in Somatic Cell Genetics, 159-187, Plenum Publ. Corp., New York (1982); Howell and Lee, Somat. Cell Molec. Genet., 15:237-244 (1989)). These mutations can result in reduced activity of respiratory complexes, especially Complex I (Howell and Nalty, Somat. Cell Molec. Genet., 14:185-193 (1988)), presumably due to decreased mitochondrial translation.
Analysis of the mitochondrial translation products of lymphoblastoid cell lines derived from MERRF patients have revealed a reduction in the labeling of the high molecular weight polypeptides relative to the smaller polypeptides (Wallace, et al., "Maternally Inherited Diseases of Man", Achievements and Perspective of Mitochondrial Research, Vol. 2, Elsevier Science Publishing, New York (1986)). This analysis has led to the speculation that MERRF may be the product of a mutation in a mtDNA, rRNA or tRNA gene.
Therefore, there exists a need to establish the molecular cause of MERRF. Prior to this invention, no method was known for the molecular diagnosis of MERRF or associated diseases. Also, there exists a need to provide a specific molecular test to accurately diagnose the presence of or susceptibility to the disease in a patient.